ATP-dependent ligases in trypanothione
biosynthesis – kinetics of catalysis and inhibition
by phosphinic acid pseudopeptides
Sandra L. Oza
1
, Shoujun Chen
2
, Susan Wyllie
1
,
James K. Coward
2
and Alan H. Fairlamb
1
1 Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, UK
2 Departments of Medicinal Chemistry and Chemistry, University of Michigan, Ann Arbor, MI, USA
Chagas’ disease, African sleeping sickness and leish-
maniasis (cutaneous, mucocutaneous and visceral) are
neglected diseases afflicting millions of people world-
wide. All of the drugs used to treat these neglected dis-
eases suffer from deficiencies such as poor efficacy,
drug resistance, toxicity or high cost of treatment [1].
The parasitic protozoa causing these diseases belong to
the order Kinetoplastida, and comparative genomic
and biochemical studies have revealed a number of
unique metabolic pathways that are being exploited
for drug discovery [2]. One of these involves trypano-
thione [N
1
,N
8

dative stress in the Kinetoplastida. The kinetic mechanism for glutathionyl-
spermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS)
obeys a rapid equilibrium random ter-ter model with kinetic constants
K
GSH
= 609 lm, K
Spd
= 157 lm and K
ATP
= 215 lm. Phosphonate and
phosphinate analogues of glutathionylspermidine, previously shown to be
potent inhibitors of GspS from Escherichia coli, are equally potent against
CfGspS. The tetrahedral phosphonate acts as a simple ground state ana-
logue of glutathione (GSH) (K
i
$ 156 lm), whereas the phosphinate
behaves as a stable mimic of the postulated unstable tetrahedral intermedi-
ate. Kinetic studies showed that the phosphinate behaves as a slow-binding
bisubstrate inhibitor [competitive with respect to GSH and spermidine
(Spd)] with rate constants k
3
(on rate) = 6.98 · 10
4
m
)1
Æs
)1
and k
4
(off

of trypanothione as drug target(s) [16].
Trypanothione is synthesized in these medically
important parasites from glutathione (GSH) and sper-
midine (Spd) by a monomeric C-N ligase [trypanothi-
one synthetase (TryS), EC 6.3.1.9], in a two-step
reaction with glutathionylspermidine as an intermedi-
ate [17–20]. Both trypanothione reductase and TryS
have been shown to be essential for parasite survival
[21–25]. However, in the insect parasite, Crithidia
fasciculata, TryS forms a heterodimer with the bi-
functional glutathionylspermidine synthetase ⁄ amidase
(GspS, EC 6.3.1.8 ⁄ GspA, EC 3.5.1.78) [26]. Previous
work suggested that each biosynthetic enzyme indepen-
dently adds successive molecules of GSH to Spd to
make trypanothione [26,27]. However, recombinant
TryS from C. fasciculata (CfTryS) has been reported
subsequently to catalyse both steps of trypanothione
synthesis [28]. Although a gene for GspS has not been
identified in Trypanosoma brucei, there is a pseudogene
in Leishmania major (accession number AJ748279) [19]
and putative genes for GspS within the genomes of
Leishmania infantum (accession number AM502243)
and Trypanosoma cruzi (accession number EAN98995)
that remain to be functionally characterized. Genome
sequencing information has also highlighted the pres-
ence of GSPS in a range of enteric pathogens such as
Salmonella and Shigella [29,30]. The mechanism and
physiological function of this protein are unknown,
but in Escherichia coli it is proposed to be involved in
regulation of polyamine levels during growth [31], and

yielded K
i
values in the nanomolar range.
Proteases that catalyse the direct addition of water
to proteins or peptides proceed via an unstable tetrahe-
dral intermediate. These enzymes are inhibited by
phosphorus-based stable mimics of the intermediate
[40]. Such high-affinity analogues are termed transition
state analogues or intermediate analogues [41]. Simi-
larly, ATP-dependent ligases involve attack of a nucle-
ophilic substrate on an electrophilic acyl phosphate
[42] via a tetrahedral intermediate. These ligases are
inhibited by stable analogues of this intermediate [43–
45]. Original work on this type of analogue based on
glutathionylspermidine was carried out on EcGspS
[46,47]. These studies investigated GSH–Spd conju-
gates (Fig. 1), with the objective of developing enzyme
inhibitors that block the biosynthesis of trypanothione
[46–51]. The synthetase activity of EcGspS was inhib-
ited by a phosphonate tetrahedral mimic, in a noncom-
petitive, time-independent manner with K
i
$ 10 lm
[47], and more potently by the phosphinate analogue
in a time-dependent manner with K
i
*
=8nm [46,50].
In each case, the phosphorus-based pseudopeptide had
no effect on the amidase activity.

–1
)
0
5
10
[Spd]
1000
500
125
250
62.5
[ATP]
[Spd] = 1000 µ
M
1000
500
125
250
62.5
500
125
250
31.25
62.5
[
S
pd] = 1000 µ
M
[ATP]
[ATP] = 500 µ

2
4
6
1/Rate (s)
0
1
2
3
1/Rate (s)
0
1
2
3
1/Rate (s)
0
1
2
1/Rate (s)
[Spd]
[ATP] = 500 µ
M
1000
500
125
250
62.5
1000
500
125
250

order, without affecting binding of the other substrates,
to form a quaternary complex, enzyme–GSH–ATP–
Spd. When a = b = c = 1, the equilibrium dissocia-
tion constants for the binding of substrate to the free
enzyme are 609 ± 26, 157 ± 5 and 215 ± 8 lm for
GSH, Spd and ATP, respectively, and k
cat
= 22.8 ±
0.6 s
)1
. When GSH and ATP were varied in a constant
ratio (10 : 1) versus various concentrations of Spd,
they produced a series of Lineweaver–Burk plots that
clearly converged (Fig. 3). This indicates that a product
release step does not occur between the binding of ATP
or GSH and Spd. Thus, the proposed kinetic model
for GspS is consistent with a random ter-reactant
mechanism, as shown in Fig. 4A.
Inhibition by phosphonate analogue
The compounds used in this study were designed to
mimic the unstable tetrahedral intermediate formed
during GspS-catalysed synthesis of glutathionylspermi-
dine (Fig. 1). However, as reported for EcGspS [47],
no time-dependent inhibition of CfGspS was observed
with the phosphonate mimic (Fig. 5), which suggests
that this compound is not acting as a mimic of the
unstable intermediate, but as a bisubstrate analogue
[56] incorporating key functional groups of both GSH
and Spd in the inhibitor. This compound behaves as a
modest classical linear competitive inhibitor of GspS

obs
. Values for k
obs
were
then plotted against the inhibitor concentration
(Fig. 6B). A linear dependency between [I] and k
obs
was
observed, and was fitted to Eqn (4) (Experimental
procedures) to obtain estimates for k
3
¢ and k
4
. The
progress curves used to determine the k
obs
values
were obtained at [S] ⁄ K
m
for GSH of 1.64. The rate
constant k
3
¢ (2.64 · 10
4
m
)1
Æs
)1
) was subsequently
corrected for competition by substrate, yielding

) is then obtained from the ratio of k
4
⁄ k
3
, yielding a
K
i
of 18.6 nm. To confirm the K
i
value, v
0
and v
s
obtained at different concentrations of inhibitor were
fitted to the equation v
s
= v
0
⁄ (1 + [I] ⁄ K
i
app
) by nonlin-
ear regression, yielding a K
i
app
value of 31.1 ± 2.1 nm,
and true K
i
value was calculated to be 19.0 ± 1.3 nm,
using the relationship K

the graph. Reaction rates are reported as catalytic centre activity
(s
)1
).
S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligases
FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5411
An alternative approach was used to obtain an inde-
pendent estimate of k
4
. In this method, the enzyme was
preincubated with excess inhibitor ([I] ‡ 10 [E]), and
the reaction was then initiated with substrate. Under
these conditions, a slow release of inhibitor is observed
until a steady state is reached. Provided that there is
no significant enzyme inactivation, substrate depletion
or product inhibition, this steady state should be iden-
tical to the steady state established when initiating with
enzyme [57]. High concentrations of enzyme and inhib-
itor were preincubated for 1 h to allow the system to
reach equilibrium. Subsequent dilution into a large
volume of assay mix containing saturating substrate
concentrations causes dissociation of the enzyme–
inhibitor complex with regain of activity. Under these
conditions, provided that the initial rate v
0
and the
effective inhibitor concentration are approximately
equal to zero, the rate of recovery of full enzyme activ-
ity will provide k
4

value of 1.36 ± 0.06 · 10
)3
Æs
)1
was
obtained, in excellent agreement with the value
obtained previously by varying the concentration of
phosphinate and initiating with enzyme.
Modality of inhibition
The mode of inhibition of the slow-binding phosphi-
nate was determined by examining the effect of varying
each substrate on the value of k
obs
at a fixed inhibitor
concentration [58]. For a competitive inhibitor, k
obs
decreases in a hyperbolic fashion with increasing
A
B
Fig. 4. Model of ter-reactant mechanism of
GspS catalysis and postulated slow-binding
inhibition by the phosphinate mimic. (A)
Kinetic mechanism. K
GSH
, K
Spd
and K
ATP
are
the equilibrium dissociation constants for

m
values
determined directly in the substrate matrix experiment
above. Thus, the phosphinate inhibitor behaves as a
slow-binding competitive bisubstrate inhibitor with
respect to GSH and Spd, but not ATP. The latter
observation is consistent with the hypothesis that an
electrophilic acyl phosphate is formed by reaction of
ATP and GSH. The acyl phosphate then reacts with
Spd to form an unstable tetrahedral intermediate,
which is mimicked by the stable tetrahedral phosphi-
nate inhibitor. The nucleotide is not a component of
the unstable tetrahedral intermediate, and therefore the
K
i
of 156 ± 13 µM
[I] = 50 µ
M
0 0.005 0.01 0.015
0
2
4
6
[I] = 100 µ
M
[I] = 200 µ
M
[I] = 400 µ
M
[I] = 0 µ

1000 l
M) as indicated. The lines fitted to the data points are linear
fits for each of the phosphonate concentrations denoted. The linear
regression values for all the data points are ‡ 0.997. (B) Kinetic
analysis of GspS inhibition by phosphonate. Assays with GspS
were performed in 250 lL of assay buffer with 1 m
M Spd, various
GSH concentrations (62.5–2000 l
M), various phosphonate concen-
trations (50–400 l
M) as indicated, and elevated levels of GspS
(200 n
M). The lines on the Lineweaver–Burk transformation are the
best global nonlinear fit of the data to Eqn (2) describing linear com-
petitive inhibition. Reaction rates are reported as catalytic centre
activity (s
)1
).
Time (min)
Product (µmol)
0
0.02
0.04
0.06
0.08
0.1
A
B
0
1

k
obs
values were calculated from Eqn (4), and the line predicts a
slope (k
3
¢) of 0.026 lM
)1
s
)1
.
S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligases
FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5413
phosphinate would not be expected to compete with
ATP in binding to the enzyme.
To determine whether the phosphinate is turned
over by CfGspS in the presence of ATP, the activity of
the enzyme (100 nm) was determined in the absence of
GSH or Spd plus or minus 1 lm phosphinate over
30 min. After correction for the background rate due
to auto-oxidation of NADPH and hydrolysis of ATP
in the coupling system, the net rates of endogenous
ATPase activity ($ 0.01% of k
cat
) in the presence and
absence of inhibitor are 1.4 (± 0.9) · 10
)3
and
3.0 (± 1.5) · 10
)3
Æs

i
app
values were determined and found to be 20–40-
fold less that that of CfGspS (Table 1). In all cases,
the slope factor was approximately 1, indicating simple
binding at a single site for all the enzymes tested.
Discussion
An understanding of the kinetic and chemical mecha-
nism of GspS and TryS involved in the biosynthesis of
glutathionylspermidine and trypanothione is crucial for
the design of inhibitors against these potential drug
targets. TryS is particularly challenging in this respect,
as these enzymes display pronounced high substrate
inhibition by GSH and form glutathionylspermidine as
an intermediate [17–19]. CfGspS does not display
substrate inhibition by GSH [35,60], and therefore
provides a convenient simple model for this class of
ATP-dependent C-N ligases.
The kinetic dataset for CfGspS fits best to a rapid
equilibrium random ter-ter reaction mechanism, and
definitively excludes a mechanism where either: (a)
ADP is released after phosphorylation of GSH prior
to binding of Spd; or (b) ADP is released following
formation of a phosphorylated enzyme intermediate
(ping-pong) prior to binding of GSH or Spd. In this
respect, the mechanism for CfGspS is similar to that
Time (min)
0 5 10 15
Product (µmol)
0

[Varied substrate] (mM)
0246810
k
obs
(s
–1
)
0
0.005
0.01
0.015
Fig. 8. Modality of inhibition by phosphinate analogue. The effect
of varying GSH (d), Spd (s) and ATP (
)onk
obs
was determined
at a fixed concentration of phosphinate. The hyperbolic fits were
obtained using either Eqn (5) for competitive inhibition (for GSH
and Spd) or Eqn (6) for uncompetitive inhibition (for ATP).
Kinetics and inhibition of ATP-dependent ligases S. L. Oza et al.
5414 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS
for c-glutamylcysteine synthetase from T. brucei [53].
However, unlike the case with c -glutamylcysteine syn-
thetase, we did not detect any marked influence of
prior binding of one substrate on the equilibrium dis-
sociation constants of the other substrates [that is, the
interaction factors a, b and c were all close to unity,
and statistical analysis did not favour their inclusion in
Eqn (1)] (Experimental procedures) [52].
Our results are also broadly in agreement with a

apparent, but may have been due to a cloning or PCR
error involving this S89N mutation. Nonetheless, we
now agree entirely with the report by Comini et al.
[28] that CfTryS is capable of catalysing both steps in
the biosynthesis of trypanothione from GSH and Spd.
A kinetic mechanism has not been determined for
the E. coli enzyme, but a reaction mechanism has been
proposed in which the glycine carboxylate of GSH is
initially phosphorylated by the c-phosphate of ATP to
form an acyl phosphate, and this is followed by nucleo-
philic attack of the N
1
-primary amine of Spd on the
acyl phosphate, leading to the formation of an unsta-
ble tetrahedral intermediate [46,48,49]. Structural
studies on EcGspS in complex with substrates and
inhibitors provide strong support for this model
[34]. Of particular note was the observation that the
slow-binding phosphinate inhibitor [46,50] had been
phosphorylated by ATP to form the tetrahedral phos-
phinophosphate in the active site, as previously postu-
lated [51]. In addition, a disordered domain in the
apoenzyme was observed to adopt an ordered confor-
mation over the active site when bound with substrates
or inhibitor. Our kinetic studies indicate that all three
substrates have to bind to the enzyme prior to cataly-
sis. This suggests that formation of the quaternary
complex induces closure of the lid domain over the
active site to form a catalytically competent complex,
thereby preventing access of water to hydrolyse the

mating to the intracellular physiological state (i.e. pH 7.2, 2 m
M Spd, 0.2 mM GSH and 2 mM Mg
2+
-ATP), and were initiated with 100 nM
each enzyme in the presence of various phosphinate concentrations. IC
50
values and slope factors are from the inhibition profiles determined
from Eqn (7), and K
i
app
values were determined from the tight-binding inhibition equation (Eqn 8). The errors represent the standard error of
the fit to the appropriate equation.
Inhibition constants
Enzyme
CfGspS CfTryS L. major TryS T. cruzi TryS T. brucei TryS
IC
50
(nM) 72 ± 6 1380 ± 380 650 ± 25 530 ± 20 1300 ± 50
Slope factor 1.2 ± 0.1 1.1 ± 0.3 1.1 ± 0.04 1.1 ± 0.04 1.1 ± 0.05
K
i
app
(nM) 29 ± 5 1330 ± 350 580 ± 30 490 ± 20 1200 ± 500
S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligases
FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5415
observed in this study and others [46,50] and con-
firmed in the crystal structure of this inhibitor bound
in the active site of EcGspS [34]. The glutathionyl-
spermidine phosphinate analogue is also a potent
inhibitor of TryS enzymes from L. major, T. cruzi and

panosomatid TryS enzymes are monomeric, or hetero-
dimeric in the case of Cf TryS and CfGspS. In this
case, the residues that interact between monomers in
EcGspS (black circles) are hardly conserved at all. One
other interesting difference between EcGspS and
CfGspS is that the latter enzyme has an additional 100
amino acids. The alignment in Fig. 9 highlights a num-
ber of insertions that are dispersed throughout the
sequence of CfGspS. These include an insertion of 17
amino acids in the amidase domain and two in the
synthetase domain (one of 14 amino acids and the
Fig. 9. Conservation of key functional residues identified for EcGspS in CfGspS and TryS. The GenBank ⁄ EMBL ⁄ DDBJ accession numbers
used to generate the alignment using
T-COFFEE are: EcGspS (U23148), CfGspS (U66520), CfTryS (AF006615), L. major TryS (AJ311570),
T. cruzi TryS (AF311782) and T. brucei TryS (AJ347018). Absolutely conserved residues are marked in bold; coloured residues indicate side
chain interactions in EcGspS with substrates or inhibitors [33]. Green triangles, residues involved in binding Mg
2+
; red triangles, three of four
residues involved in binding ATP; blue triangles, four of five residues interacting with GSH; yellow triangles, two of three residues implicated
in binding of the Spd moiety of the phosphinate inhibitor; black triangles, nonproductive binding mode, where GSH forms a mixed disulfide
with Cys338 and an isopeptide bond between the glycine moiety of GSH and Lys607 of the protein; black circles, residues that interact
between monomers in EcGspS. Only the relevant C-terminal region of the synthetase domain is shown.
Kinetics and inhibition of ATP-dependent ligases S. L. Oza et al.
5416 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS
other of 39 amino acids). It may be that these addi-
tional insertions in CfGspS are required for its hetero-
dimeric interactions with CfTryS.
From the above analysis, it is not immediately obvi-
ous why the phosphinate inhibitor is $ 20-fold less
potent against the TryS enzymes than against CfGspS

Recombinant GspS was prepared using a 60 L fermenter,
and purified to greater than 98% homogeneity as described
previously [35], except that a HiLoad Q Sepharose 16 ⁄ 10
column (GE Healthcare, Amersham, UK) was used in the
final step. Active fractions were pooled, buffer was
exchanged into 100 mm (K
+
) Hepes containing 0.01%
sodium azide, 1 mm dithiothreitol and 1 mm EDTA, and the
sample concentration was determined using the calculated
extinction coefficient of 99 370 at 280 nm. Aliquots of GspS
were then flash frozen and stored in aliquots at )80 °C.
Expression and purification of TryS enzymes
TryS enzymes from T. brucei, L. major and T. cruzi were
prepared as described previously [17–19]. In addition, we
were able to obtain functionally active CfTryS by generat-
ing a new construct in a modified pET15b vector in which
the thrombin cleavage site had been replaced by a TEV
protease cleavage site. The ORF was PCR amplified from
C. fasciculata genomic DNA using the sense primer 5¢-
CAT ATG GCG TCC GCT GAG CGT GTG CCG G-3¢,
which includes an NdeI site (underlined) and a start codon
(in bold), and the antisense primer 5¢-
GGA TCC TTA CTC
ATC CTC GGC GAG CTT G-3¢, which includes a stop
codon (in bold) and a BamHI site (underlined); the PCR
product was subsequently cloned, via pCR-Blunt II-TOPO
(Invitrogen, Paisley, UK), into the NdeI ⁄ BamHI site of
pET15bTEV. Sequencing of three independent clones
revealed that the sequence was almost identical to the

pyruvate kinase (both cou-
pling enzymes were from rabbit muscle, and purchased from
Roche), with varying amounts of ATP, GSH and Spd in a
total volume of 1 mL. Rates are expressed in moles of sub-
strate utilized per second per mole of enzyme. To determine
the kinetic mechanism, data were collected for GspS at a
range of substrate concentrations. A complete matrix of
rates as a function of substrate concentration (ATP, 31.25–
500 lm; GSH, 62.5–1000 lm; and Spd 62.5–1000 lm) was
gathered, so that for any given concentration of any one sub-
strate the rates were measured over the entire range of the
remaining two substrates. When fixed concentrations of each
of these substrates were used, the final concentrations for
ATP, GSH and Spd were 0.5, 1 and 1 mm respectively,
unless otherwise stated. The assay was initiated by adding
GspS (300 nm) and, after a lag of 10 s, the linear decrease in
absorbance was monitored for up to 1 min. Data were then
globally fitted by nonlinear regression to all possible models
for rapid equilibrium ter-reactant systems [52]. The goodness
of fit for each model was compared statistically using the
F-test and kinetic constants obtained by fitting to Eqn (1):
S. L. Oza et al. Kinetics and inhibition of ATP-dependent ligases
FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS 5417
This equation describes a rapid equilibrium random ter-ter
system, where K
GSH
, K
Spd
and K
ATP

For time-dependent inhibition by the phosphinate ana-
logue, the progress curves at different inhibitor concentra-
tions can be described by Eqn (3):
P½¼v
s
t þ½ðv
0
À v
s
Þð1 À e
Àkt
Þ=k
obs
ð3Þ
where [P] is the product concentration at time t, v
0
and v
s
are the initial and final steady-state rates, and k
obs
is the
apparent first-order rate constant for the establishment of
the final steady-state equilibrium. The resulting values for
k
obs
were plotted as a function of inhibitor concentration,
I, and fitted to Eqn (4) to obtain estimates of k
3
¢ and k
4

coupled assay described above.
To determine the modality of inhibition by the phosphi-
nate, assays were carried out in a reaction mixture of 1 mL
containing 1 lm inhibitor in addition to the other compo-
nents of the coupled assay. When GSH was varied, ATP
and Spd were kept constant at 2 and 10 mm respectively;
when Spd was varied, ATP and GSH were kept constant at
2 and 10 mm respectively; and when ATP was varied, GSH
and Spd were kept constant at 1 mm. The reaction mix was
left for 5 min at 25 °C, and the reaction was then initiated
with CfGspS (20 nm) and monitored for 15 min. These
data were then fitted to the appropriate equation [58] for
either competitive inhibition (Eqn 5)
k
obs
¼
k
1 þð½S=K
m
Þ
ð5Þ
or uncompetitive inhibition (Eqn 6)
k
obs
¼
k
1 þðK
m
=½SÞ
ð6Þ

In this equation, s is a slope factor. The equation
assumes that y falls with increasing [I]. The K
i
app
values of
t
t
V
max
¼
GSH
½
Spd
½
ATP
½
abcK
GSH
K
Spd
K
ATP
1 þ
GSH
½
K
GSH
þ
Spd
½

aK
Spd
K
ATP
þ
GSH
½
Spd
½
ATP
½
abcK
GSH
K
Spd
K
ATP
ð1Þ
Kinetics and inhibition of ATP-dependent ligases S. L. Oza et al.
5418 FEBS Journal 275 (2008) 5408–5421 ª 2008 The Authors Journal compilation ª 2008 FEBS
the inhibitor against each enzyme were determined using
the following tight-binding inhibition equation [41] (Eqn 8),
where the enzyme concentration [E] was fixed at 100 nm:
v
i
v
0
¼ 1 À
ð½Eþ½IþK
app

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